Deoxy-D-xylulose 5-phosphate (DXP) is a common precursor of thiamin (vitamin B1), pyridoxyl (vitamin B6) and isoprenoids. Isoprenoids encompass a large family of biomolecules, including vitamins A, D, E and K, cholesterol, plant pigments such as carotenoids and the phytol chain of chlorophyll, natural rubber, many essential oils, plant hormones (gibererellins, abscisic acid), insect juvenile hormone, dolichols, quinone electron carriers in mitochondria and chloroplasts, such as ubiquinone and plastoquinone, structural components of membranes (phytosterols) and Ras protein. In higher plants and bacteria, the first step in the formation of isopentenyl diphosphate, the common precursor of all isoprenoids, by the mevalonate-independent pathway is the formation of DXP from the precursors pyruvate and glyceraldehyde 3-phosphate (FIG. 1). The reaction is catalyzed by the enzyme deoxyxylulose 5-phosphate synthase (DXPS) (Lange et al. (1998) Proc Natl Acad Sci 95:2100-2104; Lois et. al. (1998) Proc Natl Acad Sci 95:2105-2110; Sprenger et al. (1997) Proc Natl Acad Sci 94:12857-12862).
The DXPS genes or cDNAs from E. coli (GenBank AF035440), Hemophilus influenzae (Swiss-Prot P54205), Rhodobacter capsulatus (Swiss-Prot P26242), Synechocystus sp. PCC6803 (GenBank D90903), Bacillus subtilis (Swiss-Prot P54523), Helicobacter pylori (GenBank AE000552), Mycoplasma tuberculosis (GenBank Z96072), Glycine max (GenBank AW278762), Lycopersicon esculentum (GenBank AF143812), Catharanthus roseus (GenBank AJ011840), Mentha x peperita (GenBank AF019383) and Arabidopsis thaliana (GenBank AF010383 and 5281015) have been cloned. Also, ESTs encoding fragments of DXPS have been identified in Oryza sativa, Ricinus communis, and Pinus taeda. However, no homologues of the DXPS genes have been identified in animals.
Disruption of the DXPS gene in Arabidopsis results in an albino phenotype due to a lack of chlorophyll and carotenoid pigments. These results indicate that DXPS is essential for chloroplast function (Lange et al.) and that inhibitors of DXPS activity may have use as herbicides. Accordingly, it would be useful to have a DXPS assay that is amenable to high throughput screening of herbicide candidates.
Several assays for DXPS activity have been reported in the literature. These assays are based on detection of the product, DXP. In these assays, the conversion of [2-.sup.14 C]-pyruvate to [.sup.14 C]-DXP in the presence of glyceraldehyde 3-phosphate and DXPS was measured by detecting [.sup.14 C]-DXP using either reverse phase HPLC (Lange, et. al.) or thin layer chromatography (Lois et al.). However, neither format is suitable for high throughput screening.